Hybrid Quinoline-Thiosemicarbazone Therapeutics as a New Treatment Opportunity for Alzheimer’s Disease‒Synthesis, In Vitro Cholinesterase Inhibitory Potential and Computational Modeling Analysis

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the leading cause of dementia worldwide. The limited pharmacological approaches based on cholinesterase inhibitors only provide symptomatic relief to AD patients. Moreover, the adverse side effects such as nausea, vomiting, loss of appetite, muscle cramps, and headaches associated with these drugs and numerous clinical trial failures present substantial limitations on the use of medications and call for a detailed insight of disease heterogeneity and development of preventive and multifactorial therapeutic strategies on urgent basis. In this context, we herein report a series of quinoline-thiosemicarbazone hybrid therapeutics as selective and potent inhibitors of cholinesterases. A facile multistep synthetic approach was utilized to generate target structures bearing multiple sites for chemical modifications and establishing drug-receptor interactions. The structures of all the synthesized compounds were fully established using readily available spectroscopic techniques (FTIR, 1H- and 13C-NMR). In vitro inhibitory results revealed compound 5b as a promising and lead inhibitor with an IC50 value of 0.12 ± 0.02 μM, a 5-fold higher potency than standard drug (galantamine; IC50 = 0.62 ± 0.01 μM). The synergistic effect of electron-rich (methoxy) group and ethylmorpholine moiety in quinoline-thiosemicarbazone conjugates contributes significantly in improving the inhibition level. Molecular docking analysis revealed various vital interactions of potent compounds with amino acid residues and reinforced the in vitro results. Kinetics experiments revealed the competitive mode of inhibition while ADME properties favored the translation of identified inhibitors into safe and promising drug candidates for pre-clinical testing. Collectively, inhibitory activity data and results from key physicochemical properties merit further research to ensure the design and development of safe and high-quality drug candidates for Alzheimer’s disease.


Introduction
Alzheimer's disease (AD), a chronic neurodegenerative disorder, is the leading cause of senile dementia. The typical symptoms include the memory dysfunction, cognitive impairment, psychiatric and behavioral abnormality, and difficulty in performing everyday tasks [1][2][3]. This multifaceted neurodegenerative disorder is one of the leading causes of death in elderly people and continues to be a social, health and economic burden on society. The exact molecular mechanism for the pathogenesis of AD is not well-understood yet; however, several hypotheses have been proposed explaining the initiation of neurodegeneration in Alzheimer's disease. These include cholinergic hypothesis (pathological changes and the dysfunction of the neuro-cholinergic system), amyloid hypothesis (β-amyloid tangles and aggregations inducing neural apoptosis, tau protein hyperphosphorylation forming senile plaque), oxidative stress hypothesis (neuro-inflammation and increasing level of reactive oxygen radicals), and bio-metal hypothesis (deregulation of transition bio-metals in AD patients). Among these, the design and development of new and potent inhibitors based on central cholinergic hypothesis remains the most common and clinically tested strategy for AD therapy [4][5][6][7][8].
Cholinesterase (ChE), namely acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8), catalyze the hydrolysis of cholinergic neurotransmitters. Acetylcholine (ACh) is predominantly decomposed by AChE compared to BuChE, thus the inhibition of AChE to increase the level of ACh remains a promising strategy for the treatment of Alzheimer's disease [9,10]. The crystal structure of this enzyme reveals the presence of a catalytic active site (CAS) and a peripheral anionic site (PAS) linked through a 20 Å long gorge. Furthermore, the role of AChE in the induction of AD through the proaggregation activity of the Aβ protein, formation of reactive oxygen species (ROS), calcium dysregulation, and neuronal dysfunction has been observed. Therefore, bioactive molecules with a potential to interact specifically with both catalytic site (PAS or CAS) residues can significantly help in the inhibition of AChE while eliminating Aβ aggregation [10].
The current treatment strategy provides only a symptomatic relief to AD patients. The previously approved (marketed) drugs, including tacrine, donepezil, rivastigmine, and galantamine, despite being diverse in structural features and pharmacokinetic profiles, are proving ineffective as medications in stopping or reversing the progression of AD [11]. Although, after 20 years, the approval of Aducanumab, treating the possible cause of the neurodegenerative disorder, rather than just the symptoms, provides a hope against this intractable condition [12], and the design of new and safer therapeutics to address multifactorial disease remains a promising research field. Therefore, employing a wellknown pharmacophore hybridization strategy could prove effective in exerting a beneficial role in the treatment of AD [1,13].

Synthetic Chemistry
The synthesis of quinoline-thiosemicarbazone hybrids 5a-k was achieved using a facile and efficient multistep approach, as illustrated in Scheme 1. Several commercially available (un)substituted anilines were acetylated using orthophosphoric acid and acetic acid to afford acetanilides 2a-f [45]. Subsequent Vilsmeier-Haack formylation using dimethylformamide and phosphoryl chloride provided 2-chloroquinoline-3-carbaldehydes 3a-f [55]. Acid-catalyzed condensation of formylquinolines with commercially available Cognizant of the fact that both quinoline and thiosemicarbazone pharmacophores are promising candidates for the generation of molecular and structural libraries of anticholinesterase inhibitors, and taking inspiration from the tacrine scaffold that features a quinoline ring, we herein utilized a pharmacophore hybridization strategy to design and explore the wider chemical space for new and potent cholinesterase inhibitors with less side effects, extending on our previous efforts in the current area of research [39][40][41][42][43][44][45]. Furthermore, in view of high demand and omnipresence of nitrogen heterocycles in numerous drugs [46][47][48][49], we have introduced an additional morpholine ring of high therapeutic value [50][51][52][53][54] in the target structures to examine the effect on the cholinesterase inhibition ( Figure 1). We have also calculated the ADME properties and the results were remarkably good.

Synthetic Chemistry
The synthesis of quinoline-thiosemicarbazone hybrids 5a-k was achieved using a facile and efficient multistep approach, as illustrated in Scheme 1. Several commercially available (un)substituted anilines were acetylated using orthophosphoric acid and acetic acid to afford acetanilides 2a-f [45]. Subsequent Vilsmeier-Haack formylation using dimethylformamide and phosphoryl chloride provided 2-chloroquinoline-3-carbaldehydes 3a-f [55]. Acid-catalyzed condensation of formylquinolines with commercially available N-(2-morpholinoethyl)hydrazinecarbothioamide 4a and N-phenylhydrazinecarbothioamide 4b gave the desired quinoline-thiosemicarbazone hybrids 5a-e in excellent yields. e, signals for morpholine ring carbon atoms appeared around 57.0 and 66.0 ppm. The ethyl chain between the morpholine and thioamide group showed peaks near 40.8 and 53.5 ppm, however the former carbon signals were overlapped by NMR solvent signal (DMSO-d6). Overall, proton integration and appropriate number of carbon signals in 1 H and 13 C NMR showed complete agreement with the corresponding structures. Furthermore, the elemental analyses of all the quinoline-thiosemicarbazones were in accordance with the proposed structures. Scheme 1. Synthetic route to quinoline-thiosemicarbazone hybrids 5a-k.

In Vitro Cholinesterase Inhibition and Structure-Activity Relationship Analyses
The newly prepared quinoline-thiosemicarbazone hybrids 5a-k were screened in vitro for their ability to inhibit cholinesterase enzymes (AChE and BChE) using Ellman's method [56]. Galantamine was employed as a positive control. The results of inhibitory Scheme 1. Synthetic route to quinoline-thiosemicarbazone hybrids 5a-k.

Spectroscopic Characterization
The condensation reaction of formylquinolines 3a-f with thiosemicarbazides 4a or 4b affording quinoline-thiosemicarbazones 5a-k was confirmed through 1 H NMR spectroscopy where the target products feature a distinct singlet for azomethine (N=CH) proton (8.26-8.61 ppm). Two additional signals attributable to secondary thioamide protons, out of which =N-NH proton resonated at a relatively more downfield chemical shift as a singlet peak at 11.76-12.23 ppm compared to C-NH proton (10.15-10.29 ppm) also confirmed the structures of thiosemicarbazones 5f-k. However, in case of compounds 5a-e, C-NH proton displayed a comparatively upfield triplet signal (8.54-8.70 ppm) due to coupling with the adjacent methylene protons. Moreover, the disappearance of a very distinct aldehyde peak around 10 ppm also confirmed the consumption of 3a-f during the course of reaction for the formation of target compounds 5a-k.
The aromatic protons showed chemical shifts between 6.80 and 9.40 ppm according to their chemical environment, with H-4 of quinoline heterocycle appearing as the most deshielded proton. Moreover, in 1 H NMR spectra of thiosemicarbazones 5a-e, the morpholine ring protons gave two sets of signals around 2.45 and 3.60 ppm in addition to linear chain methylene protons that also exhibit a similar pattern.
The structures 5a-e were further confirmed from 13 C NMR data. The C=S carbon resonated at the highest chemical shift near 177 ppm. The signals, referring to aromatic and azomethine carbon atoms, appeared between 106 and 163 ppm. In case of compounds 5a-e, signals for morpholine ring carbon atoms appeared around 57.0 and 66.0 ppm. The ethyl chain between the morpholine and thioamide group showed peaks near 40.8 and 53.5 ppm, however the former carbon signals were overlapped by NMR solvent signal (DMSO-d 6 ). Overall, proton integration and appropriate number of carbon signals in 1 H and 13 C NMR showed complete agreement with the corresponding structures. Furthermore, the elemental analyses of all the quinoline-thiosemicarbazones were in accordance with the proposed structures.

In Vitro Cholinesterase Inhibition and Structure-Activity Relationship Analyses
The newly prepared quinoline-thiosemicarbazone hybrids 5a-k were screened in vitro for their ability to inhibit cholinesterase enzymes (AChE and BChE) using Ellman's method [56]. Galantamine was employed as a positive control. The results of inhibitory assays for target compounds are presented in Table 1. The designed molecules consist of two basic components: (i) the quinoline heterocycle with varied degree of structural features (R 1 & R 2 ), which is mainly involved in π-π stacking and π-alkyl interactions, and (ii) an acyclic thiosemicarbazone fragment bearing a suitable terminal attachment (R 3 ) in the form of an aromatic ring (phenyl) and a saturated heterocyclic ring (morpholine) linked through an aliphatic linker ( Figure 2). Various nitrogen atoms can act as hydrogen bond donor sites for establishing vital interactions with the amino acid residues in the active site of enzymes. Hence, the hybridization concept not only delivered new and diversified lead structures but also maintained significantly pharmacokinetically relevant parameters, such as molar mass. As such, various dynamic structure-activity relationship analyses could be manifested depicting the effect of functional group/substituent variation on the biological potential. assays for target compounds are presented in Table 1. The designed molecules consist of two basic components: (i) the quinoline heterocycle with varied degree of structural features (R 1 & R 2 ), which is mainly involved in π-π stacking and π-alkyl interactions, and (ii) an acyclic thiosemicarbazone fragment bearing a suitable terminal attachment (R 3 ) in the form of an aromatic ring (phenyl) and a saturated heterocyclic ring (morpholine) linked through an aliphatic linker ( Figure 2). Various nitrogen atoms can act as hydrogen bond donor sites for establishing vital interactions with the amino acid residues in the active site of enzymes. Hence, the hybridization concept not only delivered new and diversified lead structures but also maintained significantly pharmacokinetically relevant parameters, such as molar mass. As such, various dynamic structure-activity relationship analyses could be manifested depicting the effect of functional group/substituent variation on the biological potential.  The evaluation of activity results presented in Table 1 revealed that the intricate balance of substituents (R 1 , R 2 & R 3 ) is critical for the strong inhibitory effects. In general, all the tested hybrid derivatives were completely selective towards AChE and showed inhibition in the range of 0.12-60.9 μM. The presence of ethylmorpholine as R 3 on the thioamide unit was significantly preferred over phenyl ring and the derivatives bearing this moiety were identified as the lead candidates. Compound 5b showed the highest inhibitory efficacy with an IC50 value of 0.12 ± 0.02 μM, 5-fold more potent than the standard drug (galantamine; IC50 = 0.62 ± 0.01 μM). For further exploration of structure-activity relationships, various alterations were considered taking into account the substituents pattern on The evaluation of activity results presented in Table 1 revealed that the intricate balance of substituents (R 1 , R 2 & R 3 ) is critical for the strong inhibitory effects. In general, all the tested hybrid derivatives were completely selective towards AChE and showed inhibition in the range of 0.12-60.9 µM. The presence of ethylmorpholine as R 3 on the thioamide unit was significantly preferred over phenyl ring and the derivatives bearing this moiety were identified as the lead candidates. Compound 5b showed the highest inhibitory efficacy with an IC 50 value of 0.12 ± 0.02 µM, 5-fold more potent than the standard drug (galantamine; IC 50 = 0.62 ± 0.01 µM). For further exploration of structure-activity relationships, various alterations were considered taking into account the substituents pattern on different sites of the hybrid structure ( Figure 3). For instance, switching the position of methoxy group from 6-position (R 1 ) to 7-position (R 2 ) on quinoline ring led to reduced potency (5c; IC 50 = 5.53 ± 0.11 µM), however, the effect of replacing ethylmorpholine with a phenyl ring was completely detrimental (5i; IC 50 = 60.9 ± 6.57 µM). Replacement of methoxy group in 5c with a methyl substituent reinstated the activity and compound 5d showed slightly better inhibition profile than galantamine with an IC 50 value of 0.55 ± 0.01 µM. Similar trend was observed for compound 5j. Disappointingly, the introduction of a halogen substituent (Cl) as R 2 produced deleterious effect in both cases (5e & 5k); however, the compound bearing an ethylmorpholine (5e) was less affected compared to its congener (5k), which showed a sharp decrease in potency (IC 50 = 47.1 ± 1.45 µM). Further modifications to compound 5b, the lead molecule, were made while removing all the substituents at quinoline ring and the results unveiled the importance of methoxy substituent at 6-position for better in vitro inhibitory properties. The resulting compound 5a showed significant activity (IC 50 = 2.95 ± 0.24 µM), but less than lead inhibitor and standard drug ( Figure 3). Collectively, the presence of both R 1 and R 2 substituents in combination with ethylmorpholine in an interactive manner is necessary for the quinoline-thiosemicarbazone hybrids to inhibit the acetylcholinesterase with maximum efficiency.

Molecular Docking Studies
For docking studies, X-ray structures of human AChE (PDB ID: 4BDT) [57] was selected due to the high crystallographic resolution (3.10 Å ) of electric eel AChE. Molecular docking analysis of all the tested compounds was performed against AChE to explore the The synthesized hybrids 5a-k have also been investigated for their potential to inhibit butyrylcholinesterase enzyme, however, the compounds showed exclusive selectivity towards acetylcholinesterase. Against butyrylcholinesterase, all the compounds showed <50% inhibition; however, in contrast to AChE results, the modest enhancement in potency preferred the presence of an aromatic (phenyl) ring instead of ethylmorpholine and a methoxy substituent appeared to be crucial for activity. The lead example was compound 5g with an IC 50 value of 11.3 ± 0.67 µM while other derivatives 5h-k showed inhibition of BChE in the range of 16-32%. Hence, the results obtained for BChE confirmed the importance of aromatic moiety on the thioamide core, albeit low activity.

Mechanism of Inhibition
With the help of kinetics studies, the mode of action of the most potent compounds 5b and 5d was determined using acetylthiocholine iodide as a substrate. Enzyme kinetics were used to determine the mechanism of acetylcholinesterase inhibition. Lineweaver-Burk graph (reciprocal of rate of reaction 1/S and reciprocal of substrate concentration 1/V) were used for the determination of the type of inhibition and analysis of effect of inhibitor on V max and K m . The slope K m /V max of each line in the Lineweaver-Burk plot was plotted against different concentrations of substrate and chemicals to determine the value of K i .
Kinetics studies were performed on 5b and 5d with different concentrations of compounds and substrate.  The most potent compound 5b presented multiple important interactions with amino acids of active pocket like π-π T-shaped (6.23 Å ) by quinoline ring and π-sulfur (4.34 Å ) by sulfur atom in the thiosemicarbazone moiety with Trp86, the most important amino acid residue within the active pocket of AChE. Hydrogen bonding was noticed between oxygen atom and Gly122 (2.82 Å ). However, Trp439 (4.68 Å ) and Tyr449 (5.86 Å ) exhibited π-sulfur interactions with sulfur atom in the thiosemicarbazone moiety of 5b. Methoxy oxygen showed hydrogen bond with Gly122 (2.82 Å ), while chloro atom formed π-lone pair (2.80 Å ) with Tyr337. Moreover, nitrogen atom of morpholine ring exhibited attractive charge interactions (4.93 Å ) with Glu452 (Figure 5b). It was clearly noted that methoxy group did not occupied the deep cleft of catalytic site.
Another potent and selective compound 5d docked inside the AChE represented sev-

Molecular Docking Studies
For docking studies, X-ray structures of human AChE (PDB ID: 4BDT) [57] was selected due to the high crystallographic resolution (3.10 Å) of electric eel AChE. Molecular docking analysis of all the tested compounds was performed against AChE to explore the possible binding modes. An overview of the active site of AChE containing cognate ligand and all the inhibitors was presented in Figure S3. The orientation of the most potent and selective compounds 5b and 5d along with the crystallographic inhibitor huprine W were presented in the active site of AChE ( Figure 5).
Molecules 2021, 26, x FOR PEER REVIEW 9 of 23 the same position, instead showed binding with other amino acids and are slightly away from the active pocket The type of interactions between ligands (5b and 5d) and receptor (4BDT) along with the distance and atoms involved in the interactions are presented in 2D interactions in Figure S4 and Table 2. The docking studies revealed that the most potent compound 5b, having 6-methoxy at quinoline ring was responsible for the conformational changes and the best binding of compound within the active pocket of enzyme. In vitro results of all the compounds showing variable inhibition against acetylcholinesterase were justified from their binding interactions within the catalytic region of the enzyme. Moreover, the selected compounds presented negative free energy values and were found to bind with significant affinity. Taken together, results presented herein showed that quinoline morpholinoethyl hydrazine carbothioamide derivatives are promising inhibitors of acetylcholinesterase. π-π T-shaped interactions are shown as fuscia, hydrogen bond as green, π-sulfur as yellow and πalkyl interaction as light green dashed lines.  The most potent compound 5b presented multiple important interactions with amino acids of active pocket like π-π T-shaped (6.23 Å) by quinoline ring and π-sulfur (4.34 Å) by sulfur atom in the thiosemicarbazone moiety with Trp86, the most important amino acid residue within the active pocket of AChE. Hydrogen bonding was noticed between oxygen atom and Gly122 (2.82 Å). However, Trp439 (4.68 Å) and Tyr449 (5.86 Å) exhibited π-sulfur interactions with sulfur atom in the thiosemicarbazone moiety of 5b. Methoxy oxygen showed hydrogen bond with Gly122 (2.82 Å), while chloro atom formed π-lone pair (2.80 Å) with Tyr337. Moreover, nitrogen atom of morpholine ring exhibited attractive charge interactions (4.93 Å) with Glu452 (Figure 5b). It was clearly noted that methoxy group did not occupied the deep cleft of catalytic site.
Another potent and selective compound 5d docked inside the AChE represented several important interactions. These include π-π T-shaped (5.43 Å) by the methyl quinoline ring, hydrogen bond (3.30 Å), by the sulfur atom of thiosemicarbazone moiety and πcation interactions (5.26 Å), and by the nitrogen atom of morpholine ring with Trp86. Additionally, the nitrogen atom of morpholine ring was involved in the formation of the salt bridge (2.69 Å) andπ-cation (4.78 Å), and interactions with Glu452 and Tyr449, respectively. Moreover, multiple π-alkyl (5.14, 5.37 and 3.76 Å) interactions were formed by methyl group with Tyr337, Tyr124 and Tyr341, respectively. The chloro group at the quinoline ring showed π-alkyl interactions (5.43 and 3.32 Å) with Trp439 and Tyr337, respectively, as shown in Figure 5c. Both compounds exhibited mostly similar interactions with the same residues, except for the presence of 6-methoxy and 7-methyl at the quinoline ring. These multiple interactions, especially π-π and strong hydrogen bonds by the potent compounds deep inside the catalytic cleft of acetylcholinesterase, can be the possible reason for their significant inhibitory profile.
The binding poses of all the derivatives were shown in Figure S3, which clearly represents the binding of compound 5b and 5d near cognate ligand showing interactions with important residues as Huprine W does. However, other residues do not show binding at the same position, instead showed binding with other amino acids and are slightly away from the active pocket The type of interactions between ligands (5b and 5d) and receptor (4BDT) along with the distance and atoms involved in the interactions are presented in 2D interactions in Figure S4 and Table 2. The docking studies revealed that the most potent compound 5b, having 6-methoxy at quinoline ring was responsible for the conformational changes and the best binding of compound within the active pocket of enzyme. In vitro results of all the compounds showing variable inhibition against acetylcholinesterase were justified from their binding interactions within the catalytic region of the enzyme. Moreover, the selected compounds presented negative free energy values and were found to bind with significant affinity. Taken together, results presented herein showed that quinoline morpholinoethyl hydrazine carbothioamide derivatives are promising inhibitors of acetylcholinesterase.

HYDE Assessment of Potent Inhibitors against Acetylcholinesterase
The top 20 docked conformations were assessed using the HYDE affinity method [58]. The assessment was made for the selected ligands using LeadIT software. The docking scores, binding free energies, and the most favorable poses for all the derivatives were calculated using FlexX ( Table 3). The results demonstrated that the potent inhibitors (5b and 5d) have high affinity towards the active site of acetylcholinesterase, as depicted by binding free energies. The potent inhibitors bind to the receptor with a very high binding affinity compared to the cognate ligands of the enzyme. Moreover, moderately and less active compounds exhibited low binding affinity as compared to potent compounds.

SeeSAR Visual Drug Design
The visual and investigative modes of the docked pose of compounds 5b and 5d revealed interpretable, innovative and important conformations using the SeeSAR tool in the LeadIT software [58,59]. Figure 6 represents the iterative and interactive optimization of leads showing the binding and non-binding capacity of compounds. Desolvation and interactions for compounds 5b and 5d are also shown in Figure 6. The approach predicts the visual and interpretable feedback for implicit hydrogen bonds and dehydration, and confirms our molecular docking results obtained using FlexX default parameters.

Molecular Dynamics Simulations
In addition to the molecular docking studies, we have performed molecular simulations for the cognate ligand (Hup W) and the most potent compound 5b showing many fold higher inhibitory activity compared to other inhibitors. The MD simulations of the enzyme in complex with the cognate ligand (Hup W) and selected inhibitor 5b were carried out in an aqueous environment for 30 ns with initial conformations taken from docking pose having the lowest binding free energy. Noncovalent interactions between ligands, Hup W, and 5b and the active site of acetylcholinesterase were monitored in a time dependent manner.
The results of MD simulations are shown in the form of RMSD values, which give information regarding the overall stability of protein and its complex with the inhibitor. As shown in Figure 7, cognate ligand (Hup W) exhibited stability from the very start soon

Molecular Dynamics Simulations
In addition to the molecular docking studies, we have performed molecular simulations for the cognate ligand (Hup W) and the most potent compound 5b showing many fold higher inhibitory activity compared to other inhibitors. The MD simulations of the enzyme in complex with the cognate ligand (Hup W) and selected inhibitor 5b were carried out in an aqueous environment for 30 ns with initial conformations taken from docking pose having the lowest binding free energy. Noncovalent interactions between ligands, Hup W, and 5b and the active site of acetylcholinesterase were monitored in a time dependent manner.
The results of MD simulations are shown in the form of RMSD values, which give information regarding the overall stability of protein and its complex with the inhibitor. As shown in Figure 7, cognate ligand (Hup W) exhibited stability from the very start soon after 5 ns and was stable for rest of the time course. The only deviation found was at 2-4.8 ns. The selected ligand (5b) showed significant stability and less deviation within the range of 0.3-0.4 nm. The little variations were noticed in the structure of apo protein, whereas the structure of complex was found significantly stable after 4 ns. The structure of 5b+protein complex showed little deviation between 14-17 ns, while remaining simulations were found stable as compared to protein alone. The consistent slight fluctuations were noted for protein during the whole simulation time (Figure 7). The radius of gyration was calculated to determine the compactness of the system during MD simulation time. It also describes the folding and unfolding of protein structure in the absence and presence of cognate ligand (Hup W) and compound 5b. The results provided in Figure 8, demonstrated the compactness of system alone and in the presence of selected cognate ligand and compound. The average scores of Rg for acetylcholinesterase and its complex with Hup W and compound 5b were found to be 2.3 and 2.25 nm, respectively, and showed the compactness of structures throughout the simulations. The results contribute towards the stability and compactness of protein only, with Hup W and compound 5b during the simulation time, therefore playing a significant role in the increased affinity of compound 5b for acetylcholinesterase enzyme.  The results recommended the overall stability of complex as compared to protein alone. The results of protein structure depicted the stability of internal motion in protein and complex systems.
The radius of gyration was calculated to determine the compactness of the system during MD simulation time. It also describes the folding and unfolding of protein structure in the absence and presence of cognate ligand (Hup W) and compound 5b. The results provided in Figure 8, demonstrated the compactness of system alone and in the presence of selected cognate ligand and compound. The average scores of Rg for acetylcholinesterase and its complex with Hup W and compound 5b were found to be 2.3 and 2.25 nm, respectively, and showed the compactness of structures throughout the simulations. The results contribute towards the stability and compactness of protein only, with Hup W and compound 5b during the simulation time, therefore playing a significant role in the increased affinity of compound 5b for acetylcholinesterase enzyme. of selected cognate ligand and compound. The average scores of Rg for acetylcholinesterase and its complex with Hup W and compound 5b were found to be 2.3 and 2.25 nm, respectively, and showed the compactness of structures throughout the simulations. The results contribute towards the stability and compactness of protein only, with Hup W and compound 5b during the simulation time, therefore playing a significant role in the increased affinity of compound 5b for acetylcholinesterase enzyme.

ADME Properties
Pharmacokinetic properties of compounds 5b and 5d were predicted to assess the impact of different parameters using previously reported prediction tools [60][61][62][63]. These parameters include molecular weight, polar surface area, number of atom types (donor/acceptor), molecular refractivity, and lipophilicity (i.e., the partition coefficient, such as log Po/w, n-octanol, WLOGP, MLOGP and XLOGP3, etc.) representing the free ener-

ADME Properties
Pharmacokinetic properties of compounds 5b and 5d were predicted to assess the impact of different parameters using previously reported prediction tools [60][61][62][63]. These parameters include molecular weight, polar surface area, number of atom types (donor/acceptor), molecular refractivity, and lipophilicity (i.e., the partition coefficient, such as log Po/w, n-octanol, WLOGP, MLOGP and XLOGP3, etc.) representing the free energies of solvation and solvent accessible surface area [64]. Moreover, water solubility predicts solubility of the compounds. These properties predict the drug-likeness and blood-brain barrier permeation of test compounds. The results presented in Table 4 suggested that the tested derivatives are safe to use as drugs. However, in case of BBB permeation, the compounds showed poor pharmacokinetics and may not be able to cross BBB, while all the other properties exhibited by selected compounds are favorable.

General
The chemicals and solvents used were of analytical grade and obtained from commercial suppliers Scharlau (Barcelona, Spain), Merck (Darmstadt, Germany), and Fluka (Buchs, Switzerland), and were used without further purification. Thin layer chromatography was performed using aluminum plates coated with silica gel 60F 254 (Merck) in an appropriate eluent. The spots were visualized using ultraviolet irradiation. Melting points were recorded on Gallenkamp melting point apparatus (UK) and were uncorrected. 1 H NMR spectra were recorded in DMSO-d 6 solvent on a Bruker Avance NMR (300 MHz, Billerica, MA, USA) spectrometer while 13

Preparation of Acetanilides 2a-f
Orthophosphoric acid (0.2 mol) was added to a stirred solution of substituted anilines 1a-f (1.0 mol) in glacial acetic acid (2.0 mol, 118 mL) and the resulting mixture was heated to reflux for 6 h. After the completion of the reaction, the mixture was poured into ice cold water with continuous stirring. The precipitated solid was filtered off, washed with excess cold water, and recrystallized from boiling water to produce acetanilides 2a-f [45].

In Vitro Cholinesterase Inhibition Assay
Inhibition of acetylcholinesterase and butyrylcholinesterase was measured in vitro by spectrophotometric method developed by Ellman [56] with slight modifications [65]. Briefly, the reaction mixture contained 60 µL phosphate buffers (KH 2 PO 4 /KOH), pH 7.7, 10 µL of test compound dissolved in DMSO (final DMSO concentration was 2%) and 10 µL of enzyme (0.5 and 3.4 U/mg of AChE or BChE, respectively). Reaction contents were mixed thoroughly and kept for 10 min during pre-incubation at 37 • C. After the pre-incubation, 10 µL of 1 mM acetythiocholine chloride or butyrylthiocholine chloride was added to the respective AChE or BChE enzyme solution to start the enzymatic reactions. DTNB (10 µL, 0.5 mM) was also added as a coloring reagent. The reaction mixture was again incubated for 20 min at 37 • C and the absorbance was measured at 405 nm using 96-well micro-plate reader. All experiments were carried out in triplicate. Galantamine (0.1 mM) was used as a standard inhibitor. In order to measure the activity of enzyme, assay was performed with a blank containing all of the components except inhibitor. The percent inhibition was calculated by the following formula: Where "A c " and "A f " are absorbance obtained for the respective enzyme (AChE and BChE) in the presence or absence of inhibitors, after subtracting the respective background (pre-read absorbance). Compounds exhibiting > 50% inhibition against ChEs were further evaluated for the determination of IC 50 values which were calculated by non-linear curve fitting program PRISM 5.0 (GraphPad, San Diego, CA, USA).

Kinetics Studies
Michaelis-Menten kinetics experiments were used to determine the type of enzyme inhibition. Detailed kinetics studies of the potent compounds 5b and 5d were performed to probe the potential mechanism of action to inhibit the enzyme. For this purpose, the initial rates of the enzyme inhibition at four concentrations of substrate (0, 0.5, 1.0, 1.5, 2.0 mM) in the absence and presence of four different concentrations of compound 5b (0, 0.06, 0.12, 0.18 µM) and compound 5d (0, 0.3, 0.6, 0.9 µM) against acetylcholinesterase were measured.

Structure Selection and Preparation
Molecular docking studies were conducted to investigate the putative interactions of the compounds making complex with the acetylcholinesterase enzyme. In order to perform docking studies, the crystallographic structure of human AChE (PDB ID: 4BDT) was obtained from the RCSB PDB database [57], and prepared for the docking analysis. Prior to the experiments, the structures of the enzyme and compounds were prepared as follows. The enzyme structure was protonated with the Protonate3D [66] algorithm implemented within the molecular modeling tool MOE [67]. The structure was energy minimized using Amber99 force field including all crystallographic solvent molecules. The backbone atoms were restrained with a small force in order to avoid collapse of the binding pockets during energy minimization calculations. Subsequently, the co-crystallized ligands and solvent molecules were removed. The crystallographic water molecules were removed and hypothetical hydrogen atoms were added to the X-ray structure in standard geometries with the MOE.

Compounds Preparation
The 3D structural coordinates of compounds were generated using MOE followed by assignment of protonation and ionization states in physiological pH range by using the "wash" module. Afterwards, the structures of compounds were energy minimized with the MMFF94x force field for docking studies.

Docking Studies
For docking studies, calculations were performed using LeadIT from BioSolveIT, GmbH Germany [68]. Receptor was loaded by Load or Prepare Receptor utility of the LeadIT software. The binding site for the receptor was defined in 9.0 Å spacing of the amino acid residues. By FlexX utility of LeadIT, docking of compounds was performed. For this purpose, compounds were docked inside the active site of receptor and 50 conformations for each ligand-receptor complex were produced based on binding free energies. Default docking parameters were not modified and top 30 highest scoring docked positions were kept for further analysis [58]. Poses with lowest free binding energy values were considered as the most stable ones having the highest affinity to interact with the receptor. Each ligandprotein complex having lowest binding free energy for interactions was examined and 3D putative binding modes were visualized using Discovery Studio Visualizer v4 [69].

Molecular Dynamics Simulations
The crystallographic structure of human AChE (PDB ID: 4BDT) [57] was obtained from the Protein Data Bank (www.pdb.org, accessed on 14 September 2021). Protein manipulation and protonation were made with the help of GROMOS96 force field having the 43a1 parameter set. The GROMACS (Groningen Machine for Chemical Simulation) simulation packages, 5.1.4 were used for the MD simulations and protocol for molecular dynamics simulations was used according to previously developed methods [70][71][72] with little modifications. Parameterization of compound 5b and Huprine W were done online using the PRODRG servers [73]. MOE and VMD [74] were used for the visualization and molecular inspection. The crystallographic structure was solvated (addition of water molecule) and counter ions were incorporated to neutralize the receptor. Subsequently, the energy minimization of the system was done, followed by equilibration using two sequential NVT (100 ps) and NPT (100 ps) runs during which protein's heavy atoms were restrained.
After minimization, the resulting ensembles were submitted to 30 ns MD simulations with a time-step of 2 fs for each simulation. Periodic boundary conditions (PBC) were applied during all the simulations. Steepest descent method was used for simple energy minimizations. All NVT and NPT runs used the Berendsen thermostat and the Parrinello-Rahman barostat for temperature (approx. 303 K) and pressure coupling (approx. 1.01 bar), respectively. The cut-off radius of 10 Å and smooth Particle Mesh Ewald (PME) protocol were observed for long-range method. The root mean square deviations, fluctuations and radius of gyration were plotted using XMGRACE v5.1.19 [75].

Conclusions
In summary, a series of new quinoline-thiosemicarbazone hybrids was designed and synthesized using a facile multistep protocol. Several commercially available anilines were successfully employed to construct quinoline heterocycle via Vilsmeyer-Haack formylation reaction. Hybridization of quinoline carbaldehydes with thiosemicarbazides afforded target compounds in excellent yields, devoiding the need of column chromatographic purification. Evaluation of cholinesterase inhibitory potential revealed the discovery of numerous potent and highly efficacious inhibitors. In particular, compound 5b inhibited the acetylcholinesterase selectively showing an IC 50 value of 0.12 ± 0.02 µM, a 5-fold high potency than galantamine (standard). Structure-activity relationship analysis showed the importance of electron-rich (methoxy) substituent at quinoline ring and ethylmorpholine on the carbothioamide unit, playing a vital role in obtaining high therapeutic efficacy. Docking, physicochemical properties, lipophilicity, water solubility, pharmacokinetics, drug-likeness, and medicinal chemistry properties were also calculated for the potent inhibitors suggesting the safer profile to be investigated as drug molecules and have high probability of blood-brain penetration and absorption. Collectively, our findings established that compound 5b is a potent, selective and competitive inhibitor of acetylcholinesterase and can serve as a promising candidate for further preclinical development for the therapy of Alzheimer's disease.
Supplementary Materials: 1 H and 13 C NMR spectra of all the synthesized compounds, dose response curves for enzyme (AChE and BChE) inhibition activity, comparative docking assessment results, 2D interactions of huprine W, compounds 5b and 5d with amino acid residues and Visual and investigative modes of the docked pose of compounds 5b and 5d within the active site of AChE are available online.